Efficient production of peptides

ABSTRACT

The present invention relates to processes for the production of peptides, and the peptides produced accordingly. Peptides produced according to the invention may be produced more efficiently than peptides produced according to prior art processes. The production process of the invention may lead to advantages in yield, purity, and/or price. Methods of marketing peptides are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/326,391, filed Jul. 8, 2014, which is acontinuation application of U.S. patent application Ser. No. 12/942,450,now U.S. Pat. No. 8,796,431, filed Nov. 9, 2010, which claims priorityfrom U.S. Provisional Application 61/259,367, filed Nov. 9, 2009, theentire contents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to processes for the production ofpeptides, and the peptides produced accordingly. Peptides producedaccording to the invention may be produced more efficiently thanpeptides produced according to prior art processes. The productionprocess of the invention may lead to advantages in yield, purity, and/orprice. Methods of marketing peptides are also disclosed.

BACKGROUND OF THE INVENTION

Peptides are becoming increasingly useful in basic research and clinicalpractice. Interest in peptides can be attributed to their role asmediators in many biological pathways and to their unique intrinsicproperties. For example, many peptides have high specificity for theirtarget with low non-specific binding to molecules that are not targeted,thus minimizing drug-drug interactions, and many peptides show lowaccumulation in tissues over time, thus reducing side effects. Moreover,peptides are often broken down in vivo to their constituent amino acids,thus reducing the risk of complications due to toxic metabolicintermediates.

Peptide market values are generally grouped within five broad categoriesin the life sciences field: cytokines, enzymes, hormones, monoclonalantibodies, and vaccines. According to a 2008 Frost & Sullivan marketresearch report, these broad categories can be further subdivided in theUnited States into vaccines (34%), monoclonal antibodies (30%),recombinant hormones and proteins (10%), gene therapy (6%), cell therapy(4%), antisense (3%), interferons (3%), interleukins (2%), growthfactors (1%), and others (7%). Each of these categories is undergoinghigh growth rates. For example, from 2004 to 2007, the monoclonalantibody market alone almost doubled its revenue from $6.3 billion to$12.6 billion. In addition, several new products, including Bydureon®(exenatide LAR for diabetes; Amylin/Eli Lilly), Extavia® (interferonbeta-1b for multiple sclerosis; Novartis), and Simponi™ (golimumab forrheumatoid and psoriatic arthritis; Centocor) have been approvedrecently by the FDA.

Moreover, peptide markets are likely to continue to grow. For example,of 633 drugs in development in 2008, nearly half were peptides. Analystsestimate an eight percent compound annual growth rate over the nexthalf-decade resulting in a potential $20 billion in earnings for theyear 2013, representing nearly fifteen percent of the forecast totalearnings for the biopharmaceuticals market. Additional opportunitiesexist for peptides as reagents in basic research and diagnosticplatforms.

While advances in the field of peptide science have led to impressivecommercial growth, several barriers remain to be overcome. For example,peptides tend to have delivery and stability problems compared totraditional small molecule therapeutics. Attempts to address theseproblems have involved oral, nasal, and pulmonary delivery. However,these attempts often require higher doses of the peptides or yieldunfavorable pharmacokinetic profiles.

One of the biggest barriers to increased use of peptides is the cost ofthe peptides themselves, which is generally significantly higher thanthe cost of producing small molecule therapeutics. For instance,Nutropin AQ®, a form of recombinant human growth hormone manufactured byGenentech and administered to patients with growth defects duringadolescence, costs $30,000 annually. High prices are an even biggerbarrier to obtaining peptides when the peptide is used for researchpurposes. For example, 3-amyloid (1-42) peptide is a research peptidewith strong implications in the onset of Alzheimer's disease. A surveyof prices from various peptide manufacturers finds the price ofresearch-grade β-amyloid (1-42) ranging from $225/mg (21^(st) CenturyBiochemicals) to $1,490/mg (Sigma-Aldrich), with the typical price at$300-$320/mg (AnaSpec, California Peptide, Innovagen, rPeptide)(estimated 2009 prices as obtained from catalogs).

Due to the importance of peptides and their high price, there is apersistent and long-felt yet unfulfilled need for lowering the cost ofpeptides. The industrial and medical use of peptides has created a needfor an improved means of production and purification, where theimprovement may be in efficiency, price, and/or quality of product.Accordingly, the present invention is directed to processes for theproduction of peptides, and the peptides produced. Methods of marketingpeptides are also disclosed.

SUMMARY OF THE INVENTION

In various embodiments, the invention is directed to a method forproducing a target peptide. According to the method of the invention, afusion peptide is produced comprising an affinity tag, a cleavable tag,and the target peptide, followed by binding of the fusion peptide to anaffinity material, cleaving the fusion peptide to release the targetpeptide; and removing the target peptide from the affinity material. Ingeneral, following binding of the fusion peptide to an affinitymaterial, the affinity material is washed to remove unbound material.Moreover, following removal of the target peptide from the affinitymaterial, the target peptide may be further modified or packaged fordistribution.

The target peptide is not limited, and may include a wide variety ofpeptides. For example, in various embodiments, the target peptide isselected from the group consisting of amyloid beta, calcitonin,enfuvirtide, epoetin, epoetin delta, erythropoietin, exenatide, factorVIII, factor X, glucocerebrosidase, glucagon-like peptide-1 (GLP-1),granulocyte-colony stimulating factor (G-CSF), human growth hormone(hGH), insulin, insulin A, insulin B, insulin-like growth factor 1(IGF-1), interferon, liraglutide, somatostatin, teriparatide, and tissueplasminogen activator (TPA). In various embodiments, the target peptideis selected from amyloid beta, enfuvirtide, exenatide, insulin, andteriparatide.

The fusion peptide may be produced in a variety of methods. In oneembodiment, the fusion peptide is produced in a bacterial expressionsystem, such as an E. coli expression system. In alternate embodiments,the expression system is a yeast expression system or a mammalianexpression system.

In various embodiments, the fusion peptide according to the inventionfurther comprises an inclusion-body directing peptide. In suchembodiments, prior to the binding of the fusion peptide to the affinitymaterial, the fusion peptide may be isolated from the expression systemby separation of inclusion bodies from the remainder of the cell in theexpression system. Following initial isolation, the fusion peptide maybe solubilized to allow further handling. In various embodiments, theinclusion-body directing peptide is selected from the group consistingof inclusion-body directing peptide is a ketosteroid isomerase, aninclusion-body directing functional fragment of a ketosteroid isomerase,an inclusion-body directing functional homolog of a ketosteroidisomerase, a BRCA2 peptide, an inclusion-body directing functionalfragment of BRCA2, or an inclusion-body directing functional homolog ofBRCA2.

In various embodiments, the affinity tag is selected from the groupconsisting of poly-histidine, poly-lysine, poly-aspartic acid, orpoly-glutamic acid. Moreover, the cleavable tag may be selected from thegroup consisting of Trp, His-Met, Pro-Met, and an unnatural amino acid.In the event of more than one cleavable tag in the fusion peptide, thevarious cleavable tags may be orthogonal, i.e. have different reactivitywith any particular cleavage agent. In various embodiments, the cleavingstep is performed with an agent selected from the group consisting ofNBS, NCS, or Pd(H2O)4.

Methods according to the invention are also directed to evaluating thecommercial market for a target peptide comprising a) producing a targetpeptide according to the methods described herein; b) making sampleamounts of the target peptide available for no cost or minimal cost; andc) measuring the number of requests for the target peptide over a periodof time.

In addition to the above methods, the invention is directed to thetarget peptide produce according to the invention, in particular totarget peptides of greater than 99% purity. Also, vectors for use inexpression systems for the production of target peptides according tothe invention are envisioned. For example, vectors according to theinvention may include a nucleotide sequence encoding an affinity tag; anucleotide sequence encoding a cleavable tag; and a nucleotide sequenceencoding a target peptide; wherein the nucleotides are arranged inoperable combination and further wherein expression of the operablecombination results in a fusion protein comprising an affinity tag, acleavable tag, and a target peptide. Additional embodiments of theinvention are directed to a cell comprising the vectors described hereinas well as a fusion protein produced according to the methods describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 diagrams a modified form of the commercially available pET-19bvector (pET-19bmhb1). Such vectors can be used to produce a KSI sequenceflanked by two NcoI restriction sites and a histidinetag-tryptophan-β-amyloid (1-42) sequence flanked by two XhoI restrictionsites.

FIG. 2 illustrates activation of transcription in a commerciallyavailable pBAD promoter via the addition of L-arabinose. Arabinose bindsto AraC (“C” in the diagram) and causes the protein to release the O₂site and bind the I₂ site which is adjacent to the I₁ site. Thisreleases the DNA loop and allows transcription to begin. A second levelof control is present in the cAMP activator protein (CAP)-cAMP complex,which binds to the DNA and stimulates binding of AraC to I₁ and I₂.Basal expression levels can be repressed by introducing glucose to thegrowth medium, which lowers cAMP levels and in turn decreases thebinding of CAP, thus decreasing transcriptional activation.

FIG. 3 shows exemplary data for gel electrophoresis of samples takenfrom a cell culture prior to induction and then 2, 4, 6, and 16 hourspost-induction. Optimal fusion protein synthesis occurs when the cultureis induced and grown overnight.

FIG. 4A-D presents four embodiments of amino acid sequences forketosteroid isomerase.

FIG. 5 presents one embodiment of a nucleic acid sequence forketosteroid isomerase.

FIG. 6 shows exemplary data for the stages of inclusion body preparationby gel electrophoresis of cells lysed with high-power sonication. Thelysed material was washed with a series of buffers containing differentconcentrations of Tris, NaCl, PMSF, Triton-X100, and urea, and thesupernatant was collected. The disappearance of the 21 kD band duringsuccessive steps and reappearance of the 21 kD band upon solubilizingthe inclusion bodies (lane 10) indicates that the inclusion bodies wereproperly prepared.

FIG. 7 illustrates one embodiment of an immobilized Ni-NTA resin bindingto a 6×His tag on a protein.

FIG. 8 presents exemplary data showing gel electrophoresis followingNi-NTA affinity chromatography. Inclusion bodies were loaded onto anequilibrated Ni-NTA column and washed with the same buffer, collectingthe flow-through (lane 1). The column was then washed with 50% EtOH asto equilibrate it with the cleavage solution buffer (lane 2). On-columncleavage was performed with 3×NBS for 30 minutes at room temperature andthe flow through was collected (lane 3). The column was washed with 300mM imidazole to wash off all remaining fusion protein and theflow-through was collected (lane 4).

FIG. 9 illustrates one possible mechanism for the selective cleavage oftryptophan peptide bonds with NBS (N-bromosuccinimide). According to themechanism, the active bromide ion halogenates the indole ring of thetryptophan residue followed by a spontaneous dehalogenation through aseries of hydrolysis reactions. These reactions lead to the formation ofan oxindole derivative which promotes the cleavage reaction. In FIG. 9,Z-Trp-Y is cleaved at the carboxy terminus of the Trp residue to yield amodified Z-Trp and a free amino group on Y (i.e., H₂N—Y).

FIG. 10 presents exemplary data for gel electrophoresis of ninedifferent NBS cleavage reactions. The reactions were performed and thesamples were run on an 18% acrylamide gel and silver stained. Lane (1)stock inclusion bodies; lane (2) OX NCS for 0 min; lane (3) 1×NBS for 30min; lane (4) 1×NBS for 60 min; lane (5) 3×NBS for 0 min; lane (6) 3×NBSfor 30 min; lane (7) 3×NBS for 60 min; lane (8) 6×NBS for 0 min; lane(9) 6×NBS for 30 min; lane (10) 6×NBS for 60 min.

FIG. 11 presents exemplary data showing gel electrophoresis of inclusionbodies that were reacted with 3×NBS for 30 min and then quenched withN-acetylmethionine. The same sample was loaded in increasing quantitiesfrom 10 to 25 μl to show appearance of 5 kD cleavage product.

FIG. 12 presents the chemical structures of a variety of unnatural aminoacids that have been incorporated into peptides and proteins by cellsystems through genetic modification of the cell systems. See Wang, etal., (2009) Chem Biol. 16(3):323-36.

FIG. 13 presents an estimated manufacturing cost analysis of directmaterials used. Based on an average inclusion body preparation of 5grams per liter, a 50% cleavage success rate, and product loss duringpurification, the materials to synthesize beta-amyloid (1-42) cost only$0.11 per milligram.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for producing fusion peptidesthat can be purified and cleaved into desired peptides, and the peptidesproduced according to the methods. In various embodiments, the method ofthe invention includes induction, inclusion body isolation, affinitycolumn purification, and chemical cleavage. In various embodiments, thepresent invention utilizes an expression vector to make the peptidesaccording to the invention. In some aspects, by combining molecularexpression technologies that employ genetically-malleable microorganismssuch as E. coli cells to synthesize a peptide of interest withpost-expression isolation and modification, one can synthesize a desiredpeptide rapidly and efficiently. In various embodiments, the inventionconcerns production of fusion peptides that can be purified usingaffinity separation and cleaved with a chemical reagent to release atarget peptide.

In various embodiments, the invention is directed to a vector thatencodes an inclusion body targeting sequence, an affinity tag tofacilitate purification, and a specific amino acid sequence thatfacilitates selective chemical cleavage. Variously, the inclusion bodytargeting amino acid sequence comprises between 1 and 125 amino acids ofa ketosteroid isomerase protein. The affinity tag sequence may comprisea poly-histidine, a poly-lysine, poly-aspartic acid, or poly-glutamicacid. In one embodiment, the vector further comprises an expressionpromoter located on the 5′ end of the affinity tag sequence. In oneembodiment, the invention is directed to a vector that codes for aspecific sequence that facilitates selective chemical cleavage to yielda peptide of interest following purification. Such chemically cleavableamino acid sequences include Trp, His-Met, or Pro-Met.

In one embodiment, the present invention contemplates a peptideexpression vector, comprising: a) a first nucleotide sequence encodingan affinity tag amino acid sequence; b) a second nucleotide sequenceencoding an inclusion body targeting amino acid sequence; c) a thirdnucleotide sequence encoding a chemically cleavable amino acid sequence;and d) a promoter in operable combination with the first, second, andthird nucleotide sequences.

In one embodiment, the present invention contemplates a method forproducing a peptide of commercial or therapeutic interest comprising thesteps of: a) cleaving a vector with a restriction endonuclease toproduce a cleaved vector; b) ligating the cleavage site to one or morenucleic acids, wherein the nucleic acids encode a desired peptide havingat least a base overhang at each end configured and arranged forligation with the cleaved vector to produce a second vector suitable forexpression of a fusion peptide; c) transforming the second vector intosuitable host cell; d) incubating the host cell under conditionssuitable for expression of the fusion peptide; e) isolation of inclusionbodies from the host cell; f) solubilization and extraction of thefusion peptide from the inclusion bodies; g) binding of the fusionpeptide to a suitable affinity material; h) washing of bound fusionpeptide to remove impurities; and i) cleaving the fusion peptide torelease the said target peptide.

Peptides produced by the methods of the invention may have significantlylower costs and/or other advantageous features. These potentiallycheaper costs may lie not only in less expensive raw materials requiredfor synthesis, but also may lie in less chemical waste which isgenerated compared to the traditional process of solid phase peptidesynthesis, or in more efficient processing to achieve a certain purity,thus lowering the cost of the material. Furthermore, the exclusion of awaste stream may be particularly beneficial to the environment. Invarious embodiments, processes according to the invention provide a highyield of peptide with high purity. In various embodiments, peptidesproduced according to the invention may be R&D grade peptides orclinical grade therapeutics.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art.

As used herein, the term “peptide” is intended to mean any polymercomprising amino acids linked by peptide bonds. The term “peptide” isintended to include polymers that are assembled using a ribosome as wellas polymers that are assembled by enzymes (i.e., non-ribosomal peptides)and polymers that are assembled synthetically. In various embodiments,the term “peptide” may be considered synonymous with “protein,” or“polypeptide.” In various embodiments, the term “peptide” may be limitedto a polymer of greater than 50 amino acids, or alternatively, 50 orfewer amino acids. In various embodiments, the term “peptide” isintended to include only amino acids as monomeric units for the polymer,while in various embodiments, the term “peptide” includes additionalcomponents and/or modifications to the amino acid backbone. For example,in various embodiments, the term “peptide” may be applied to a corepolymer of amino acids as well as derivatives of the core polymer, suchas core polymers with pendant polyethylene glycol groups or corepolymers with amide groups at the amino or carboxy terminus of the aminoacid chain.

As used herein, “consisting essentially of” may exclude those featuresnot listed herein that would otherwise alter the operation of theinvention. However, the use of the phrase “consisting essentially of”does not exclude features that do not alter the operation of therequired components.

The term “polymer” is a molecule (or macromolecule) composed ofrepeating structural units connected by covalent chemical bonds.

A “patient,” “subject” or “host” to be treated with the composition ofthe present invention may mean either a human or non-human animal. Theterm “mammal” is known in the art, and exemplary mammals include humans,primates, bovines, porcines, canines, felines, and rodents (e.g., miceand rats).

I. Target Peptides

The methods of the invention are applicable to a wide range of peptidesas the isolated product, which may be referred to as target peptides.Peptides produced according to the methods of the invention may benaturally-occurring peptides, non-naturally-occurring peptides, ornaturally-occurring peptides with non-natural substitutions, deletions,or additions. In various embodiments, the target peptide may be modifiedchemically or biologically following isolation to yield a derivative ofthe target peptide, such as a target peptide with one or morecarboxamide groups in place of free carboxy groups.

In various embodiments, the peptide is selected from vaccines,antibodies, recombinant hormones and proteins, interferons,interleukins, and growth factors. In some embodiments, the targetpeptide is fifty or fewer amino acids. In some embodiments, the targetpeptide is greater than fifty amino acids.

Further non-limiting embodiments of the invention include peptides andanalogs thereof selected from the group consisting of angiotensin,arginine vasopressin (AVP), AGG01, amylin (IAPP), amyloid beta,N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase), avian pancreaticpolypeptide (APP), B-type natriuretic peptide (BNP), calcitoninpeptides, calcitonin, colistin (polymyxin E), colistin copolymer 1(Cop-1), cyclosporin, darbepoetin, PDpoetin, dornase alfa, eledoisin,β-endorphin, enfuvirtide, enkephalin pentapeptides, epoetin, epoetindelta, erythropoietin, exenatide, factor VIII, factor X,follicle-stimulating hormone (FSH), alpha-galactosidase A (Fabrazyme),Growth Hormone Releasing Hormone 1-24 (GHRH 1-24), β-globin, glucagon,glucocerebrosidase, glucagon-like peptide-1 (GLP-1), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), growth hormone, Hepatitis B viral envelope protein,human growth hormone (hGH), insulin, insulin A, insulin B, insulin-likegrowth factor 1 (IGF-1), interferon, kassinin, alpha-L-iduronidase(rhIDU; laronidase), lactotripeptides, leptin, liraglutide (NN2211,VICTOZA), luteinizing-hormone-releasing hormone, methoxy polyethyleneglycol-epoetin beta (MIRCERA), myoglobin, neurokinin A, neurokinin B,NN9924, NPY (NeuroPeptide Y), octreotide, pituitary adenylate cyclaseactivating peptide (PACAP), parathyroid hormone (PTH), Peptide HistidineIsoleucine 27 (PHI 27), proopiomelanocortin (POMC) peptides,prodynorphin peptides, polymyxins, polymyxin B, Pancreatic PolYpeptide(PPY), Peptide YY (PYY), secretin, somatostatin, Substance P,teriparatide (FORTEO), tissue plasminogen activator (TPA),thrombospondins (TSP), ubiquitin, urogastrone, Vasoactive IntestinalPeptide (VIP, or PHM27), and viral envelope proteins. In variousembodiments, the target peptide is selected from amyloid beta,calcitonin, enfuvirtide, epoetin, epoetin delta, erythropoietin,exenatide, factor VIII, factor X, glucocerebrosidase, glucagon-likepeptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), humangrowth hormone (hGH), insulin, insulin A, insulin B, insulin-like growthfactor 1 (IGF-1), interferon, liraglutide, somatostatin, teriparatide,and tissue plasminogen activator (TPA). In various embodiments, thetarget peptide is selected from amyloid beta and insulin.

In various embodiments, the target peptide is a hormone. For example, invarious embodiments, the target peptide is selected from the groupconsisting of Activin, inhibin, Adiponectin, Adipose derived hormones,Adrenocorticotropic hormone, Afamelanotide, Agouti signalling peptide,Allatostatin, Amylin, Angiotensin, Atrial natriuretic peptide, Bovinesomatotropin, Bradykinin, Brain-derived neurotrophic factor, CJC-1295,Calcitonin, Ciliary neurotrophic factor, Corticotropin-releasinghormone, Cosyntropin, Endothelin, Enteroglucagon, Follicle-stimulatinghormone, Gastrin, Gastroinhibitory peptide, Glucagon, Glucagon hormonefamily, Glucagon-like peptide-1, Gonadotropin, Granulocytecolony-stimulating factor, Growth hormone, Growth hormone releasinghormone, Hepcidin, Human chorionic gonadotropin, Human placentallactogen, Incretin, Insulin, Insulin glargine, Insulin lispro, Insulinaspart, Insulin-like growth factor 2, Insulin-like growth factor,Leptin, Liraglutide, Luteinizing hormone, Melanocortin,Melanocyte-stimulating hormone, Melanotan II, Minigastrin, N-terminalprohormone of brain natriuretic peptide, Nerve growth factor,Neurotrophin-3, NPH insulin, Obestatin, Orexin, Osteocalcin, Pancreatichormone, Parathyroid hormone, Peptide YY, Peptide hormone, Plasma reninactivity, Pramlintide, Preprohormone, Proislet Amyloid Polypeptide,Prolactin, Relaxin, Renin, Salcatonin, Secretin, Sincalide, Teleostleptins, Thyroid-stimulating hormone, Thyrotropin-releasing hormone,Urocortin, Urocortin II, Urocortin III, Vasoactive intestinal peptide,and Vitellogenin.

In various embodiments, the target peptide is already commerciallyavailable through a production process that differs from the processaccording to the invention. While not wishing to be bound by theory, itis believed that peptides produced according to the present inventionwill have differing levels of residual components from the process ofproduction. For example, in comparison with peptides of the samesequence produced according to conventional recombinant processes,peptides produced according to the present invention may be expected tohave fewer residual cellular contaminants upon initial purification.Alternatively, in comparison with peptides of the same sequence producedby conventional synthetic processes, peptides produced according to thepresent invention may be expected to have fewer residual chemicalcontaminants upon initial purification. Various commercially availablepeptides may be found in paper catalogs or in online catalogs, forexample, from Sigma-Aldrich(<<sigmaaldrich.com/life-science/cell-biology/peptides-and-proteins.html>>),California Peptide (<<californiapeptide.com/peptide_catalog_table>>),CPCScientific (<<cpcscientific.com/products/browseCatalog.asp>>), andBachem (<<shop.bachem.com/ep6sf/index.ep>>), the contents of each ofwhich are hereby incorporated by reference.

In various embodiments, target peptides do not include tryptophan intheir sequence.

II. Inclusion-Body Directing Peptides

Inclusion bodies are composed of insoluble and denatured forms of apeptide and are about 0.5-1.3 μm in diameter. These dense and porousaggregates help to simplify recombinant protein production since theyhave a high homogeneity of the expressed protein or peptide, result inlower degradation of the expressed protein or peptide because of ahigher resistance to proteolytic attack by cellular proteases, and areeasy to isolate from the rest of the cell due to differences in theirdensity and size relative to the other cellular components. In variousembodiments, the presence of inclusion bodies permits production ofincreased concentrations of the expressed protein or peptide due toreduced toxicity by the protein or peptide upon segregation into aninclusion body. Once isolated, the inclusion bodies may be solubilizedto allow for further manipulation and/or purification.

An inclusion-body directing peptide is an amino acid sequence that helpsto direct a newly translated protein or peptide into insolubleaggregates within the host cell. Prior to final isolation, in variousembodiments of the invention the target peptide is produced as a fusionpeptide where the fusion peptide includes as part of its sequence ofamino acids an inclusion-body directing peptide. The methods of theinvention are applicable to a wide range of inclusion-body directingpeptides as components of the expressed fusion protein or peptide.

In various embodiments, the inclusion-body directing peptide is aketo-steroid isomerase (KSI) sequence, a functional fragment thereof, ora functional homolog thereof.

In various embodiments, the inclusion-body directing peptide is a BRCA-2sequence, a functional fragment thereof, or a functional homologthereof.

III. Affinity-Tag Peptides

According to the invention, a wide variety of affinity tags may be usedAffinity tags useful according to the invention may be specific forcations, anions, metals, or any other material suitable for an affinitycolumn. In one embodiment, any peptide not possessing an affinity tagwill elute through the affinity column leaving the desired fusionpeptide bound to the affinity column via the affinity tag.

Specific affinity tags according to the invention may includepoly-lysine, poly-histidine, poly-glutamic acid, or poly-argininepeptides. For example, the affinity tags may be 5-10 lysines, 5-10histidines, 5-10 glutamic acids, or 5-10 arginines. In variousembodiments, the affinity tag is a hexahistidine sequence, hexa-lysinesequence, hexa-glutamic acid sequence, or hexa-arginine sequence.Alternatively, the HAT-tag (Clontech) may be used. In variousembodiments, the affinity tag is a His-Trp Ni-affinity tag.

Without wishing to be bound by theory, it is believed that the histidineresidues of a poly-histidine tag bind with high affinity to Ni-NTA orTALON resins. Both of these resins contain a divalent cation (Ni-NTAresins contain Mg²⁺; TALON resins contain Co²⁺) that forms a highaffinity coordination with the His tag.

In various embodiments, the affinity tag has a pI (isoelectric point)that is at least one pH unit separate from the pI of the target peptide.Such difference may be either above or below the pI of the targetpeptide. For example, in various embodiments, the target peptide has ahigh pI, and the affinity tag has a pI that is at least one pH unitlower, at least two pH units lower, at least three pH units lower, atleast four pH units lower, at least five pH units lower, at least six pHunits lower, or at least seven pH units lower. Alternatively, the targetpeptide has a low pI, and the affinity tag has a pI that is at least onepH unit higher, at least two pH units higher, at least three pH unitshigher, at least four pH units higher, at least five pH units higher, atleast six pH units higher, or at least seven pH units higher. In oneembodiment, the target peptide has a pI of about 10 and the affinity taghas a pI of about 6.

In various embodiments, the affinity tag is contained within the nativesequence of the inclusion body directing peptide. Alternatively, theinclusion body directing peptide is modified to include an affinity tag.For example, in one embodiment, the affinity tag is a KSI or BRCA2sequence modified to include extra histidines, extra lysines, extraarginines, or extra glutamic acids.

In various embodiments, epitopes may be used such as FLAG (EastmanKodak) or myc (Invitrogen) in conjunction with their antibody pairs.

IV. Cleavable Tags

The methods of the invention are applicable to a wide range of cleavabletags.

In various embodiments, the cleavable tag is a tryptophan at the aminoterminus of the target peptide. Upon cleavage with a cleaving agent, theamide bond connecting the tryptophan to the target peptide is cleaved,and the target peptide is released from the affinity column.

In various embodiments, the cleavable tag is a tryptophan at the aminoterminus of the target peptide, where the cleavable tag also includes anamino acid with a charged side-chain in the local environment of thetryptophan, such as within five amino acids on the upstream (i.e. amino)or downstream (i.e. carboxy) side of the tryptophan. In variousembodiments, the presence of an amino acid side-chain within five aminoacids on the amino terminus of the tryptophan amino acid allows forselectivity of cleavage of the tryptophan of the cleavable tag over anyother tryptophans that may be present in the fusion peptide, forexample, tryptophans as part of the inclusion body directing peptide oras part of the target peptide. For example, in various embodiments, anamino acid with a positively charged side chain such as lysine,ornithine, or arginine is within five, four, three, or two amino acidunits, or is adjacent on the amino terminus to the tryptophan of thecleavable tag. In various embodiments, a glutamic acid amino acid iswithin five, four, three, or two amino acid units, or is adjacent on theamino terminus to the tryptophan of the cleavable tag.

In various embodiments, the cleavable tag is His-Met, or Pro-Met.

In various embodiments, the cleavable tag is an unnatural amino acid.Cells have been modified to enable the cells to produce peptides whichcontain unnatural amino acids. For instance, Wang, et al., (2001)Science 292:498-500, describes modifications made to the proteinbiosynthetic machinery of E. coli which allow the site-specificincorporation of an unnatural amino acid, O-methyl-L-tyrosine, inresponse to an amber stop codon (TAG). Wang, et al., (2009) Chem Biol.16(3):323-36 provides a review of numerous unnatural amino acids thathave been site-specifically incorporated into proteins in E. coli,yeast, or mammalian cells. Without wishing to be bound by theory, it isbelieved that incorporation of one or more unnatural amino acids canprovide additional selectivity for cleavage at the unnatural amino acidover non-specific cleavage at other sites on the fusion peptide. Invarious embodiments, the unnatural amino acid is selected from compounds1-27 in FIG. 12.

In some aspects, the present invention includes the production of fusionpeptides comprising unnatural amino acids. In some aspects, prokaryoticcells with modifications to the protein biosynthetic machinery producesuch fusion peptides. Examples of such prokaryotic cells include E.coli. In some aspects the modifications comprise adding orthogonaltRNA/synthetase pairs. In some aspects four base codons encode novelamino acids. In some aspects, E. coli allow the site-specificincorporation of the unnatural amino acid O-methyl-L-tyrosine into apeptide in response to an amber stop codon (TAG) being included in anexpression vector.

V. Fusion Peptide Synthesis

Numerous methods for producing peptides may be adapted to the invention.Various methods are described in detail herein.

A. Ribosomal Synthesis

In various embodiments, peptides may be produced by ribosomal synthesis,which utilizes the fundamental methods of transcription and translationto express peptides. Ribosomal synthesis is usually performed bymanipulating the genetic code of various expression systems. Somepeptides can be expressed in their native form in eukaryotic hosts suchas Chinese hamster ovary (CHO) cells. Animal cell culture may requireprolonged growing times to achieve maximum cell density and may achievelower cell density than prokaryotic cell cultures (see Cleland, J.(1993) ACS Symposium Series 526, Protein Folding: In Vivo and In Vitro,American Chemical Society). Bacterial host expression systems such asEscherichia coli may achieve higher productivity than animal cellculture, and may have fewer regulatory hurdles for peptides intended tobe used therapeutically. Numerous U.S. patents on general bacterialexpression of recombinant proteins exist, including U.S. Pat. No.4,565,785.

In one embodiment, the expression system is a microbial expressionsystem. For example, in one embodiment, the process uses E. coli cells.

1. Construction of Vectors

In various embodiments, the method of the invention involves theconstruction of a DNA vector which includes certain selectable markers(such as antibiotic resistance in the case of E. coli) enablingselective screening against the cells that do not contain theconstructed vector with the gene of interest. Vectors according to theinvention may include hybrid promoters and multiple cloning sites forthe incorporation of different genes. Various expression vectors mayinclude the pET system and the pBAD system.

The pET system encompasses more than 40 different variations on thestandard pET vector. In various embodiments, the pET system utilizes aT7 promoter that is recognized specifically by T7 RNA polymerase. Thispolymerase can transcribe DNA five times faster than E. coli RNApolymerase allowing for increased levels of transcription. In variousembodiments, the Escherichia coli is protease deficient.

In one embodiment, a vector is designed with a sequences coding for afusion peptide comprising an inclusion-body directing peptide, anaffinity tag peptide, a cleavable peptide, and the target peptide. Forexample, in one embodiment, the vector is a pET-19b vector is modifiedto include a ketosteroid isomerase (KSI) sequence as the inclusion-bodydirecting peptide. Thus, following cleavage of a restriction site suchas the NcoI restriction site and insertion of the KSI sequence, the KSIsequence is flanked by two NcoI restriction sites. In addition, such avector may be modified to include a histidine tag sequence as thesequence coding for an affinity tag adjacent to a tryptophan-encodingtag sequence as the sequence coding for a cleavable peptide which isfurther adjacent to a sequence coding for a target peptide such as thebeta-amyloid (1-42) sequence. If an XhoI restriction site is used forpurposes of insertion, the newly inserted sequence is flanked by twoXhoI restriction sites. FIG. 1 diagrams one embodiment of a modifiedpET-19b (pET-19bmhb1) vector that can be used to produce a KSI sequenceflanked by two NcoI restriction sites, and a histidinetag-tryptophan-beta-amyloid (1-42) sequence flanked by two XhoIrestriction sites.

As such, a vector according to the invention such as a modified pET-19bvector contains the desired fusion peptide in a four part sequence: aKSI sequence or functional fragment to sequester the synthesized fusionprotein into inclusion bodies, an affinity tag such as hexahistidine, acleavage tag such as a tryptophan, and the target peptide.

2. Inoculation and Induction or Activation

Upon construction of an appropriate vector, the vector may be introducedinto a host cell according to any method, and expression of the desiredfusion peptide may be induced or activated by any method in the art.

In some embodiments once constructed, a vector according to theinvention is inoculated or transformed into competent cells. In variousembodiments, the competent cells may be mammalian cells such as Chinesehamster ovary cells, or microbial cells, such as E. coli cells. Forexample, the cells may be commercially available, such as DH5-ot E. colicells (available from Invitrogen).

In various embodiments, transformed cells can be plated onto agarcontaining an antibacterial agent to prevent the growth of any cellsthat do not contain a resistance gene, thereby selecting for cells thathave been transformed. In some embodiments, transformed E. coli cellsare plated onto agar containing ampicillin to prevent the growth of anyE. coli strains that do not contain the constructed pET-19b vector, anda colony is selected for further expansion.

Colonies from the plating process may be grown in starter culture orbroth according to standard cell culture techniques. For example, insome embodiments, one colony from an agar plate is grown in a starterculture of broth, which may optionally contain an antibacterial agent.Typically, cells are grown to a preselected optical density before beingfurther processed to obtain fusion peptide. For example, cells may begrown to an optical density (OD) of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, or 0.9, all values being about. In some embodiments the cells aregrown to an optical density (OD) of about 0.5.

In a bacterial expression system, once the vector-containing bacterialcells have been isolated, inducible transcription may be used to producethe desired fusion peptide. For example, in E. coli cells, the lacoperon serves as an inducible promoter that is activated under certainenvironmental conditions. E. coli are always capable of metabolizing themonosaccharide glucose. However, in order to metabolize the disaccharidelactose, the cells need an enzyme known as β-galactosidase. Thus, lowextracellular glucose concentrations and high lactose concentrationsinduce the lac operon and the gene for β-galactosidase is transcribed.Accordingly, in various embodiments, an inducible promoter such as thelac operon is situated upstream from the sequence coding for the fusionpeptide. Upon induction of the lac operon, transcription of the sequencecoding for the desired fusion peptide occurs.

The term “activation” refers to the removal of repressor protein. Arepressor protein is generally allosteric meaning it changes shape whenbound by an inducer molecule and dissociates from the promoter. Thisdissociation allows for the transcription complex to assemble on DNA andinitiate transcription of any genes downstream of the promoter.Therefore, by splicing genes produced in vitro into the bacterialgenome, one can control the expression of novel genes. This trait may beused advantageously when dealing with inclusion bodies if the productionand amassing of inclusion bodies becomes toxic enough to kill E. coli.For example, expression of the desired fusion peptide can be delayeduntil a sufficient population of cells has been cultured, and then thepromoter can be induced to express a large amount of fusion peptide byremoval of the repressor protein. Thus, the L-arabinose operon may beactivated according to the invention for increased protein expression ata desired timepoint. Specifically, the L-arabinose operon may beactivated by both the addition of L-arabinose into the growth medium andthe addition of IPTG, a molecule that acts as an activator to dissociatethe repressor protein from the operator DNA. FIG. 2 illustrates oneembodiment of the activation of transcription in a pBAD vector via theaddition of L-arabinose. Without wishing to be bound by theory, it isbelieved that L-arabinose binds to the AraC dimer causing the protein torelease the O₂ site on the DNA and bind to the I₂ site. These stepsserve to release the DNA loop and enable its transcription.Additionally, the cAMP activator protein (CAP) complex stimulates AraCbinding to I₁ and I₂—a process initiated with IPTG.

3. Targeting Expressed Peptides to Inclusion Bodies

In some cases, cells expressing only a fusion peptide with an affinitytag, a cleavable tag, and the target peptide cannot produce largeamounts of fusion peptide. The reasons for low production yields mayvary. For example, the fusion peptide may be toxic to the bacteria, thuscausing the bacteria to die upon production of certain levels of thefusion peptide. Alternatively, the target peptide may be either poorlyexpressed or rapidly degraded in the bacterial system. In variousscenarios, the target peptide may be modified by the host cell,including modifications such as glycosylation. To remedy some or all ofthese problems, the desired fusion peptide may be directed to aninclusion body, thereby physically segregating the target peptide fromdegradative factors in the cell's cytoplasm or, in the case of targetpeptides that are toxic to the host such as peptide antibiotics,physically segregating the target peptide to avoid toxic effects on thehost. Moreover, by physically aggregating the fusion peptide in aninclusion body, the subsequent separation of the fusion peptide from theconstituents of the host cell and the media (i.e., cell culture orbroth) may be performed more easily or efficiently.

Target peptides may be directed to inclusion bodies by producing thetarget peptide as part of a fusion peptide where the target peptide islinked either directly or indirectly via intermediary peptides with aninclusion-body directing peptide. In various embodiments, an otherwiseidentical fusion peptide without an inclusion-body directing peptide hasminimal or no tendency to be directed to inclusion bodies in anexpression system. Alternatively, an otherwise identical fusion peptidewithout an inclusion-body directing peptide has some tendency to bedirected to inclusion bodies in an expression system, but the number,volume, or weight of inclusion bodies is increased by producing a fusionpeptide with an inclusion-body directing peptide. In variousembodiments, where the target peptide itself directs the fusion peptideof the invention to inclusion bodies, a separate inclusion-bodydirecting peptide may be excluded.

Any inclusion-body directing peptide may be used according to themethods of the invention. For example, methods have been described whichallow α-human atrial natriuretic peptide (α-hANP) to be synthesized instable form in E. coli. Eight copies of the synthetic α-hANP gene werelinked in tandem, separated by codons specifying a four amino acidlinker with lysine residues flanking the authentic N and C-termini ofthe 28 amino acid hormone. That sequence was then joined to the 3′ endof the fragment containing the lac promoter and the leader sequencecoding for the first seven N terminal amino acids of β-galactosidase.The expressed multidomain protein accumulated intracellularly intostable inclusion bodies and was purified by urea extraction of theinsoluble cell fraction. The purified protein was cleaved into monomersby digestion with endoproteinase lys C and trimmed to expose theauthentic C-terminus by digestion with carboxypeptidase B. See Lennicket al., “High-level expression of α-human atrial natriuretic peptidefrom multiple joined genes in Escherichia coli,” Gene, 61:103-112(1987), incorporated by reference herein.

In various embodiments, directing the target peptide to an inclusionbody by producing the target peptide as part of a fusion peptide maylead to higher output of peptide. For example, in various embodiments,the desired fusion peptide is produced in concentrations greater than100 mg/L. In various embodiments, the desired fusion peptide is producedin concentrations greater than about 200 mg/L, 250 mg/L, 300 mg/L, 350mg/L, 400 mg/L, 450 mg/L, 500 mg/L, 550 mg/L, 600 mg/L, 650 mg/L, 700mg/L, 750 mg/L, 800 mg/L, 850 mg/L, 900 mg/L, 950 mg/L, and 1 g/L, allamounts being prefaced by “greater than about.” In various embodiments,the output of desired fusion peptide is greater than about 1.5 g/L,greater than about 2 g/L, or greater than about 2.5 g/L. In variousembodiments, the output of desired fusion peptide is in the range offrom about 500 mg/L to about 2 g/L, or from about 1 g/L to about 2.5g/L. In one embodiment, the desired fusion peptide is produced in yieldsequal to or greater than 500 mg/L of media.

In one embodiment, the inclusion-body directing peptide is a ketosteroidisomerase (KSI) or inclusion-body directing functional fragment thereof.In certain embodiments, inclusion-body directing functional fragment hasat least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, or at least 100amino acids. Homologs of a ketosteroid isomerase are also encompassed.Such homologs may have at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, or atleast 95 percent sequence identity with the amino acid sequence of aketosteroid isomerase. In various embodiments, an expression system fora fusion peptide with a functional fragment or homolog of a ketosteroidisomerase will produce at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, or greater than 100 percent of the amount of inclusion bodiesproduced by an otherwise identical expression system with a fusionpeptide containing a complete ketosteroid isomerase peptide sequence.

B. Solid Phase Peptide Synthesis

In one embodiment, the desired fusion peptide is made through solidphase peptide synthesis (SPPS). SPPS involves covalently linking a shortpeptide to an insoluble polymer providing a structural support for theelongation of the peptide. To achieve elongation of the peptide, thepractitioner performs a series of repeated cycles de-protecting thechemically reactive portions of amino acids, linking the de-protectedfree terminal amine (N) to a single N-protected amino acid,de-protecting the N-terminal amine of the newly added residue, andrepeating this process until the desired peptide has been built.Additional measures may be necessary for peptides that are about 50 ormore amino acids in length.

In one embodiment, the solid phase peptide synthesis uses Fmocprotecting groups. The Fmoc protecting group utilizes a base labilealpha-amino protecting group. In an alternative embodiment, the solidphase peptide synthesis uses Boc protecting groups. The Boc protectinggroup is an acid labile alpha-amino protecting group. Each method mayinvolve distinct resin addition, amino acid side-chain protection, andconsequent cleavage/deprotection steps. Generally, Fmoc chemistrygenerates peptides of higher quality and in greater yield than Bocchemistry. Impurities in Boc-synthesized peptides are mostly attributedto cleavage problems, dehydration and t-butylation. Once assembled onthe solid support, the peptide is cleaved from the resin using stronglyacidic conditions, usually with the application of trifluoracetic acid(TFA). It is then purified using reverse phase high pressure liquidchromatography, or RP-HPLC, a process in which sample is extrudedthrough a densely packed column and the amount of time it takes fordifferent samples to pass through the column (known as a retention time)is recorded. As such, impurities are separated out from the sample basedon the principle that smaller peptides pass through the column withshorter retention times and vice versa. Thus, the protein being purifiedelutes with a characteristic retention time that differs from the restof the impurities in the sample, thus providing separation of thedesired protein.

Solid-phase peptide synthesis generally provides high yields becauseexcess reagents can be used to force reactions to completion. Separationof soluble byproducts is simplified by the attachment of the peptide tothe insoluble support throughout the synthesis. Because the synthesisoccurs in the same vessel for the entire process, mechanical loss ofmaterial is low.

In various embodiments, an inclusion body directing peptide may beexcluded. Alternatively, an inclusion body directing peptide may beincluded to provide beneficial folding properties and/orsolubility/aggregating properties.

C. Non-Ribosomal Synthesis

In various embodiments, peptides may be produced by non-ribosomalsynthesis. Such peptides include circular peptides and/or depsipeptides.

Nonribosomal peptides are synthesized by one or more nonribosomalpeptide synthetase (NRPS) enzymes. These enzymes are independent ofmessenger RNA. Nonribosomal peptides often have a cyclic and/or branchedstructure, can contain non-proteinogenic amino acids including D-aminoacids, carry modifications like N-methyl and N-formyl groups, or areglycosylated, acylated, halogenated, or hydroxylated. Cyclization ofamino acids against the peptide backbone is often performed, resultingin oxazolines and thiazolines; these can be further oxidized or reduced.On occasion, dehydration is performed on serines, resulting indehydroalanine.

The enzymes of an NRPS are organized in modules that are responsible forthe introduction of one additional amino acid. Each module consists ofseveral domains with defined functions, separated by short spacerregions of about 15 amino acids. While not wishing to be bound bytheory, it is thought that a typical NRPS module is organized asfollows: initiation module, one or more elongation modules, and atermination module. The NRPS genes for a certain peptide are usuallyorganized in one operon in bacteria and in gene clusters in eukaryotes.

In various embodiments, an inclusion body directing peptide may beexcluded. Alternatively, an inclusion body directing peptide may beincluded to provide beneficial folding properties and/orsolubility/aggregating properties.

VI. Separation of Fusion Peptide from Formation Media

Following production of the desired fusion peptides, separation from theproduction media is required. Optionally, following separation, thedesired fusion peptide and carrier may be concentrated to remove excessliquid. Numerous methods for separating fusion peptides from theirformation media and subsequent handling may be adapted to the invention.Various methods are described in detail herein.

A. Fusion Peptides Targeted to Inclusion Bodies

In various embodiments, the cells used to produce the desired fusionpeptides may be lysed to release the fusion peptides. For example, wherethe desired fusion peptide is aggregated in inclusion bodies, the cellmay by lysed, followed by separation of the inclusion bodies from theproduction media and cellular detritus. Any method of cell lysis may beused according to the invention.

In various embodiments, cells are disrupted using high-power sonicationin a lysis buffer. For example, a lysis buffer may be added before lysiscontaining Tris, sodium chloride, glycerol, and a protease inhibitor. Inone embodiment, a lysis buffer containing about 25 mM Tris pH 8.0, about50 mM NaCl, about 10% glycerol, and the protease inhibitor 1000×PMSF mayadded before lysis. Insoluble inclusion bodies may be collected usingone or more washing steps and centrifugation steps. Wash buffers mayinclude any reagents used for the stabilization and isolation ofproteins. For example, in various embodiments, wash buffers are usedcontaining varying concentrations of Tris pH 8.0, NaCl, and Triton X100.

In an embodiment of the invention, targeting the desired fusion peptideto an inclusion body may result in higher initial purity upon lysis ofthe cell. For example, in one embodiment, lysis of the cell andisolation of inclusion bodies through physical means such ascentrifugation may result in an initial purity of greater than about70%, great than about 75%, greater than about 80%, greater than about85%, greater than about 90%, or greater than about 95% for the desiredfusion peptide.

In some embodiments following cell lysis, inclusion bodies form a pelletand remain in the pellet rather than supernatant until a solubilizationstep. In various embodiments, the pellet is washed clean of theremaining cellular components, and insoluble inclusion bodies aresolubilized in a buffer for further handling. Solubilization buffers mayinclude urea or any other chaotropic agent necessary to solubilize thefusion peptide. Without wishing to be bound by theory, it is believedthat the solubilization step involves solubilizing the inclusion bodiesin a chaotropic agent which serves to disrupt the peptides byinterfering with any stabilizing intra-molecular interactions.

In various embodiments, the solubilization buffer may include urea,guanidinium salts, or organic solvents. For example, a solubilizationbuffer may contain about 25 mM Tris pH 8.0, about 50 mM, NaCl, about 0.1mM PMSF, and about 8M urea. In some embodiments, solubilization ofinclusion bodies occurs with the addition of 8M urea as the solechaotropic agent, and other chaotropic agents are excluded.Alternatively, the solubilization buffer may exclude urea or guanidiniumsalts. For example, in one embodiment, guanidinium salts are excluded toavoid interference with further processing on an ion exchange column. Asan additional example, in one embodiment, high urea concentrations suchas about 8M urea are excluded to avoid denaturing proteases that may beincluded in the solubilization buffer.

In various embodiments, a minimal amount of solubilization buffer isused. In the event that excess solubilization buffer is present, thesolution may be processed to remove excess solvent prior to furtherpurification.

B. Fusion Peptides not Targeted to Inclusion Bodies

In various embodiments, fusion peptides are not directed to inclusionbodies. In such embodiments, the fusion peptides may remain in thecytosol of the cell, or the fusion peptides according to the inventionmay be secreted from the cell. Soluble fusion peptides may be isolatedby any method, such as centrifugation, gel electrophoresis, pH or ionexchange chromatography, size exclusion chromatography, reversed-phasechromatography, dialysis, osmosis, filtration, and extraction.

VII. Purification by Affinity Chromatography

Following cell lysis and initial isolation and solubilization of fusionpeptides according to the invention, the fusion peptides are furtherpurified by affinity chromatography, which is a highly selective processthat relies on biologically-relevant interactions between an immobilizedstationary phase and the fusion peptide to be purified. In variousembodiments, the immobilized stationary phase is a resin or matrix.Without wishing to be bound by theory, it is believed that affinitychromatography functions by selective binding of the desired componentfrom a mixture to the immobilized stationary phase, followed by washingof the stationary phase to remove any unbound material.

According to the invention, a wide variety of affinity chromatographysystems may be used. For example, polyhistidine binds with greataffinity and specificity to nickel and thus an affinity column ofnickel, such as QIAGEN nickel columns, can be used for purification.See, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology10.11.8 (John Wiley & Sons 1993). Alternatively, Ni-NTA affinitychromatography resin (available from Invitrogen) may be used. FIG. 7provides a schematic of an example of an immobilized Ni-NTA resinbinding to a 6×HisTag on a protein. Metal affinity chromatography hasbeen used as a basis for protein separations. See Arnold, “MetalAffinity Separations: A New Dimension In Protein Processing”Bio/Technology, 9:151-156 (1991). See also Smith et al., “ChelatingPeptide-immobilized Metal Ion Affinity Chromatography” J. Biol. Chem.,263:7211-7215 (1988), which describes a specific metal chelating peptideon the NH₂ terminus of a protein that can be used to purify that proteinusing immobilized metal ion affinity chromatography.

According to the invention, the affinity column is equilibrated withbuffer which may be the same as used for the solubilization of thefusion peptide. The column is then charged with the solubilized fusionpeptide, and buffer is collected as it flows through the column. Invarious embodiments, the column is washed successively to remove ureaand/or other impurities such as endotoxins, polysaccharides, andresidual contaminants remaining from the cell expression system.

VIII. Removal of Target Peptide from Affinity Column Via Cleavage

Numerous methods for cleavage of the fusion peptides on the affinitycolumn may be adapted to the invention. In general, the cleavage stepoccurs by introduction of a cleavage agent which interacts with thecleavage tag of the fusion peptide resulting in cleavage of the fusionpeptide and release of the target peptide. Following cleavage, theaffinity column may be flushed to elute the target peptide while theportion of the fusion peptide containing the affinity tag remains boundto the affinity column. Following elution of the target peptide, theeluting solution may be condensed to a desired concentration. The targetpeptide may be further processed and/or packaged for distribution orsale.

Control of the cleavage reaction may occur through chemical selectivity.For example, the cleavage tag may include a unique chemical moiety whichis absent from the remainder of the fusion peptide such that thecleavage agent selectively interacts with the unique chemical moiety ofthe cleavage tag.

In various embodiments, control of the cleavage reaction occurs througha unique local environment. For example, the cleavage tag may include achemical moiety that is present elsewhere in the fusion peptide, but thelocal environment differs resulting in a selective cleavage reaction atthe cleavage tag. For example, in various embodiments, the cleavage tagincludes a tryptophan and a charged amino acid side chain within fiveamino acids of the tryptophan. In various embodiments, the charged aminoacid is on the amino terminus of the tryptophan amino acid.

In various embodiments, control of the cleavage reaction may occurthrough secondary or tertiary structure of the fusion peptide. Forexample, in various embodiments, where identical moieties are present inthe cleavage tag and elsewhere in the fusion peptide, the other portionsof the fusion peptide may fold in secondary or tertiary structure suchas alpha-helices, beta-sheets, and the like, to physically protect thesusceptible moiety, resulting in selective cleavage at the cleavage tag.

In various embodiments, minor or even major differences in selectivityof the cleavage reaction for the cleavage tag over other locations inthe fusion peptide may be amplified by controlling the kinetics of thecleavage reaction. For example, in various embodiments, theconcentration of cleavage agent is controlled by adjusting the flow rateof eluting solvent containing cleavage agent. In various embodiments,the concentration of cleavage agent is maintained at a low level toamplify differences in selectivity. In various embodiments, thereservoir for receiving the eluting solvent contains a quenching agentto stop further cleavage of target peptide that has been released fromthe column.

Moreover, various methods for removal of peptides from affinity columnsmay be excluded. For example, methods according to the invention mayspecifically exclude the step of washing an affinity column with asolution of a compound with competing affinity in the absence of acleavage reaction. In one embodiment, the step of washing an affinitycolumn with a solution of imidazole as a displacing agent to assist inremoving a fusion peptide from an affinity column is specificallyexcluded.

In various embodiments, multiple cleavages are envisioned. For example,insulin is known to be produced from a proinsulin precursor requiringtwo cleavage events. Both cleavage events are required in order for themature insulin to be properly folded. Accordingly, in variousembodiments, the process according to the invention may include twocleavage tags. Preferably, when more than one cleavage tag is present,the distinct cleavage tags are orthogonal, or able to be cleaved withspecificity by different cleavage agents. For example, in oneembodiment, one cleavage tag is a methionine amino acid while the othercleavage tag is a tryptophan amino acid.

In various embodiments, the cleavage agent is selected from the groupconsisting of NBS, NCS, cyanogen bromide, Pd(H₂O)₄, 2-ortho iodobenzoicacid, DMSO/sulfuric acid, or a proteolytic enzyme. Various methods andcleavage agents are described in detail herein.

A. NBS Cleavage

In one embodiment, the cleavage reaction according to the inventioninvolves the use of a mild brominating agent N¬bromosuccinimde (NBS) toselectively cleave a tryptophanyl peptide bond at the amino terminus ofthe target peptide. Without wishing to be bound by theory, it isbelieved that in aqueous and acidic conditions, NBS oxidizes the exposedindole ring of the tryptophan side chain, thus initiating a chemicaltransformation that results in cleavage of the peptide bond at thissite. FIG. 9 illustrates one possible mechanism for the selectivecleavage of tryptophan peptide bonds with N-bromosuccinimde. Accordingto the mechanism, the active bromide ion halogenates the indole ring ofthe tryptophan residue followed by a spontaneous dehalogenation througha series of hydrolysis reactions. These reactions lead to the formationof an oxindole derivative which promotes the cleavage reaction.

B. NCS Cleavage

In one embodiment, the cleavage reaction according to the inventioninvolves the use of a mild oxidizing agent N¬chlorosuccinimde (NCS) toselectively cleave a tryptophanyl peptide bond at the amino terminus ofthe target peptide. Without wishing to be bound by theory, it isbelieved that in aqueous and acidic conditions, NCS oxidizes the exposedindole ring of the tryptophan side chain, thus initiating a chemicaltransformation that results in cleavage of the peptide bond at thissite.

C. Enzymatic Cleavage

In various embodiments, enzymes may be employed to cleave the fusionprotein. For example, Protease use is diverse yet selective as there aremany proteases that recognize specific amino acid sequences. In variousembodiments, the active site of a serine or threonine protease will bindto either serine or threonine, respectively, and intitiate catalyticmechanisms that result in proteolysis. Additional enzymes includecollagenase, enterokinase factor X_(A), thrombin, trypsin, clostripainand alasubtilisin. See Uhlen and Moks, Meths. in Enz., 185:129-143(1990) and Emtage, “Biotechnology & Protein Production” in DeliverySystems for Peptide Drugs, pp. 23-33 (1986).

D. Additional Chemical Agents

In various embodiments, the cleavage agent is a chemical agent such ascyanogen bromide, palladium (II) aqua complex (such as Pd(H₂O)₄), formicacid, and hydroxylamine. For example, cyanogen bromide may be used toselectively cleave a fusion peptide at a methionine amino acid at theamino terminus of the target peptide.

IX. Downstream Processing

In various embodiments, target peptides produced according to theprocess of the invention may be further modified. For example, invarious embodiments, the C-terminus of the target peptide is connectedto alpha-hydroxyglycine. At the desired time, the target peptide, eitheras the isolated target peptide or as part of the fusion peptide, isexposed to acid catalysis to yield glycolic acid and a carboxamide groupat the carboxy terminus of the target peptide. A carboxamide group atthe carboxy terminus is present in a variety of neuropeptides, and isthought to increase the half-life of various peptides in vivo.

In various embodiments, target peptides produced according to theinvention may be further modified to alter in vivo activity. Forexample, in various embodiments, a polyethylene glycol (PEG) group maybe added to a target peptide.

X. Peptide Marketing

Methods according to the invention are also directed to marketing thetarget peptides produced according to the methods of the invention. Forexample, in one embodiment, the invention is directed a method ofevaluating the commercial market for a target peptide. Such methods mayinclude producing a target peptide as described herein, making sampleamounts of the target peptide available for no cost or for minimal cost,and measuring the number of requests for the target peptide over aperiod of time. Advantages of making a target peptide available in thismanner may include an improved calculation of the future supplies neededand/or future demand by paying customers. Alternatively, providing atarget peptide at no cost or minimal cost initially may induce interestin the target peptide and the discovery of favorable characteristics forthe peptide that spur future sales. Minimal cost may include a pricethat is approximately the cost of production with essentially no profitinvolved. In various embodiments, the minimal cost may be about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% ofthe price of a competitor's product.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

EXAMPLES

The examples herein provide some non-limiting examples according to theinvention. The following examples include both actual examples andprophetic examples.

Example 1

Cells were induced to initiate the synthesis of KSI-Abeta (1-42) with 1mM IPTG (Invitrogen) and 0.2% L-arabinose (Calbiotech) as follows.Plated cells are incubated overnight at 37° C. and then one colony fromthis plate was grown up overnight in a starter culture of 8 mL of Luriabroth+ampicillin. The following morning, the starter culture wasinoculated into 1 L of Luria broth+ampicillin and grown to an opticaldensity (OD) of 0.5. At this point, the cells were induced with 1 mMIPTG (Invitrogen) and 0.2% L-arabinose (Calbiotech) to initiate thesynthesis of KSI-Abeta (1-42).

To optimize the amount of KSI-Abeta (1-42) production in the bacteria,samples of the 1 L inoculation were taken prior to inducing thebacteria, and then 2, 4, 6, and 16 hours (overnight growth) afterinduction. A 12% acrylamide gel was used to analyze the samples sincethe fusion protein weighs approximately 21 kD. FIG. 3 shows exemplarydata for gel electrophoresis of samples taken from a cell culture priorto induction and then 2, 4, 6, and 16 hours post-induction. Optimalfusion protein synthesis occurred when the culture was induced and grownovernight.

Eight hours after induction, the cells were re-induced with the sameconcentrations of IPTG and L-arabinose as well as 100 mg of ampicillinas to prevent the growth of any new strains of E. coli.

Example 1A

The construct was re-designed to place a His-tag upstream from the KSIsequence rather than downstream.

Example 2

Following induction of KSI-Abeta (1-42) production in E. coli, lysisbuffer containing 25 mM Tris pH 8.0, 50 mM NaCl, 10% glycerol, and theprotease inhibitor 1000×PMSF was added before lysis. Insoluble inclusionbodies were collected using washing and centrifugation. Three differentwash buffers were used containing varying concentrations of Tris pH 8.0,NaCl, and Triton X100. Once washed clean of the remaining cellularcomponents, the insoluble inclusion bodies were solubilized in a buffercontaining 25 mM Tris pH 8.0, 50 mM, NaCl, 0.1 mM PMSF, and 8M urea. The8M urea served as a chaotropic agent necessary in solubilizing protein.

A 12% acrylamide gel was run on both uninduced and induced bacteria, thecell lysate produced from high output sonication, and the supernatantfrom each washing step during the inclusion body preparation. The gelwas stained with Coomassie Blue reagent. The appearance of a 21 kD inthe induced sample provides evidence for inclusion body synthesisresulting from induction. FIG. 6 presents exemplary data showing thestages of inclusion body preparation by gel electrophoresis of cellslysed with high-power sonication and washed with a series of bufferscontaining different concentrations of Tris, NaCl, PMSF, Triton-X100,and urea. The disappearance of the 21 kD band during successive stepsand reappearance of the 21 kD band upon solubilizing the inclusionbodies (lane 10) indicated that the inclusion bodies were properlyprepared. Accordingly, the lane containing the cell lysate was almostentirely blue because as the cells are ruptured, relatively largequantities of various proteins were extracted. As the lysate was washedrepeatedly of impurities, the lanes became clearer.

Example 3

The concentration of protein in solubilized inclusion bodies wasdetermined via a Bradford Assay. A series of NBS cleavage reactions wasrun to determine the optimal conditions for tryptophanyl peptide bondcleavage. Three concentrations of NBS purchased from TCI America(equimolar, 3×, and 6×) were allowed to react with KSI-Abeta (1-42) forvarying amounts of time (0, 15, and 30 minutes) before being quenchedwith excess N-acetylmethionine (Acros). Since the amyloid beta cleavageproduct weighed only 5 kD, a higher percentage acrylamide gel (18%) wasused to determine the success of the NBS cleavage in solution. The gelindicated that optimal cleavage occurred when 6×NBS was reacted withKSI-Abeta (1-42) at room temperature from 0 to 30 minutes. FIG. 10presents exemplary data for gel electrophoresis of nine different NBScleavage reactions. The samples were run on an 18% acrylamide gel andsilver stained. Lane (1) stock inclusion bodies; lane (2) OX NCS for 0min; lane (3) 1×NBS for 30 min; lane (4) 1×NBS for 60 min; lane (5)3×NBS for 0 min; lane (6) 3×NBS for 30 min; lane (7) 3×NBS for 60 min;lane (8) 6×NBS for 0 min; lane (9) 6×NBS for 30 min; lane (10) 6×NBS for60 min.

Example 4

Ni-NTA Affinity Chomatography resin purchased from Invitrogen wasequilibrated with the same solubilization buffer as in the inclusionbody preparation. Next, the resin was charged with the solubilizedinclusion bodies and the flow through was collected. The column was thenwashed with five column volumes of 50% EtOH to remove urea and flowthrough. Afterwards, 3×NBS was loaded and the column was placed on arocker for 30 minutes. At this time, the reaction was quenched withexcess N-acetylmethionine and the flow through was collected. The columnwas then washed with 300 mM imidazole to discharge the remaining fusionprotein and the flow through was collected.

SDS-PAGE analysis indicated that a very small amount of inclusion bodiesadhere to the Ni-NTA column as evidenced by the appearance of a large 21kD band in the first wash. FIG. 8 presents exemplary data for gelelectrophoresis following Ni-NTA affinity chromatography as follows.Inclusion bodies were loaded onto an equilibrated Ni-NTA column andwashed with the same buffer, collecting the flow-through (lane 1). Thecolumn was then washed with 50% EtOH as to equilibrate it with thecleavage solution buffer (lane 2). On-column cleavage was performed with3×NBS for 30 minutes at room temperature and the flow through wascollected (lane 3). The column was washed with 300 mM imidazole to washoff all remaining fusion protein and the flow-through was collected(lane 4). A narrower band appeared after the second wash in EtOH toequilibrate the column for the on-column cleavage. A very minor amountof cleavage did occur on the remaining KSI-Abeta (1-42). Incubating theinclusion bodies overnight on a rocker did not improve on-columncleavage, although it did improve the initial binding of KSI-Abeta(1-42) to the column.

Example 5

SDS-PAGE analysis on the NBS solution cleavage indicated that thecleavage was successful in solution. FIG. 11 presents exemplary datashowing gel electrophoresis of inclusion bodies that were reacted with3×NBS for 30 min and then quenched with N-acetylmethionine. The samesample was loaded in increasing quantities (from 10 to 25 μl) to showappearance of 5 kD cleavage product.

Because optimal cleavage rates range from 35-45 percent, only a smallamount of KSI-Abeta (1-42) was produced. Since the gel contained a smallamount of diluted sample, the assay did not detect the 5 kD cleavageproduct with Coomassie Blue staining. Therefore, visualizing thecleavage product required overloading the sample and overdeveloping thesilver stain. See, FIG. 11, faint appearance of the 5 kD band in the tworightmost lanes.

Example 6

The manufacturing cost analysis of direct materials used indicated thatthe cost to synthesize beta-Amyloid (1-42) through a combination ofrecombinant expression, chemical manipulation, and purification isdrastically lower than the prices charged by other manufacturers (notingthat a large makeup of their prices contains overheads, operating costs,labor costs, etc.). Assuming an average yield of inclusion bodies for a1 liter culture is approximately 5 grams per liter and a 50% NBScleavage rate, as well as considering product lost during purification,an estimated $0.11 in materials is all that is necessary to synthesize 1mg of beta-Amyloid (1-42). Even after factoring all of the othermanufacturing costs such as facilities, equipment, and labor into theprice, the synthesis of beta-Amyloid (1-42) using this method could costmuch less to the consumer. See FIG. 12.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

1.-22. (canceled)
 23. A method for producing a target peptide, themethod comprising: expressing a heterologous fusion peptide in agenetically modified cell, the heterologous fusion peptide comprising anaffinity tag, a cleavable tag, and the target peptide, wherein thecleavable tag is tryptophan (Trp), and wherein the affinity tag or thecleavable tag is heterologous to the target peptide; binding theheterologous fusion peptide to an affinity material via the affinitytag; and cleaving the heterologous fusion peptide with DMSO while boundto the affinity material to release the target peptide, therebyproducing the target peptide.
 24. The method of claim 23, wherein thetarget peptide of the heterologous fusion peptide is expressed in nativeform.
 25. The method of claim 23, further comprising the step ofsolubilizing the heterologous fusion peptide after the step ofexpressing a heterologous fusion peptide.
 26. The method of claim 23,wherein the target peptide is selected from amyloid beta, calcitonin,enfuvirtide, epoetin, epoetin delta, erythropoietin, exenatide, factorVIII, factor X, glucocerebrosidase, glucagon-like peptide-1 (GLP-1),granulocyte-colony stimulating factor (G-CSF), human growth hormone(hGH), insulin, insulin A, insulin B, insulin-like growth factor 1(IGF-1), interferon, liraglutide, somatostatin, teriparatide, or tissueplasminogen activator (TPA).
 27. The method of claim 23, wherein thestep of expressing a heterologous fusion peptide in a geneticallymodified cell is performed in a bacterial expression system.
 28. Themethod of claim 27, wherein the bacterial expression system is anEscherichia coli expression system.
 29. The method of claim 23, whereinthe step of expressing a heterologous fusion peptide in a geneticallymodified cell is performed in a yeast expression system.
 30. The methodof claim 23, wherein the heterologous fusion peptide further comprisesan inclusion-body directing peptide.
 31. The method of claim 28, whereinprior to binding the heterologous fusion peptide to an affinitymaterial, the method further comprises removal of inclusion bodiescontaining the heterologous fusion peptide from the Escherichia coliexpression system and solubilization of the heterologous fusion peptide.32. The method of claim 28, wherein the inclusion-body directing peptideis selected from a ketosteroid isomerase or a BRCA2 peptide.
 33. Themethod of claim 23, wherein subsequent to binding the heterologousfusion peptide to the affinity material, the method further compriseswashing the affinity material to remove unbound material.
 34. The methodof claim 23, wherein the affinity tag is selected from poly-histidine,poly-lysine, poly-aspartic acid, or poly-glutamic acid.
 35. The methodof claim 23, wherein the target peptide is present in its native formafter releasing the target peptide.
 36. The method of claim 23, whereinthe heterologous fusion peptide is secreted from the cell afterexpressing the heterologous fusion peptide.
 37. The method of claim 23,further comprising lysing the cell after expressing the heterologousfusion peptide.
 38. The method of claim 23, wherein the target peptidethat is produced is greater than 95% pure.
 39. The method of claim 23,wherein the target peptide that is produced is greater than 99% pure.40. The method of claim 23, wherein the target peptide is refolded afterthe target peptide is released.
 41. The method of claim 23, wherein therate of cleavage of the cleavable tag is modified by adjusting theconcentration of the DMSO.